Anti-angiogenic therapy represents an emerging and promising modality for the treatment of malignancies, although controlled comparisons between putative angiogenesis inhibitors has been lacking. Gene therapy approaches are well suited for the experimental delivery of diverse anti-angiogenic proteins, given the ease of vector construction versus recombinant protein, and the convenience of single injection viral administration. We have exploited an adenoviral approach to create an array of adenoviruses expressing anti-angiogenic proteins such as soluble ectodomains of the Vascular Endothelial Growth Factor (VEGF) receptors Flk 1, Flt 1 and neuropilin, as well as the anti-angiogenic factors endostatin and angiostatin. Using these viruses, we have performed the first comparison between VEGF-based and non-VEGF-based anti-angiogenic proteins, and have found superior antiangiogenic and anti-tumor activity with soluble Flk 1 and Flt 1 treatment over tumor-derived proteins such as endostatin and angiostatin. The soluble FIk 1 and Flt 1 adenoviruses exhibit broad-spectrum suppression of numerous human and murine tumors in both subcutaneous and orthotopic models. The current proposal extends upon these findings in several respects. First, we will explore the hypothesis that VEGF blockade can be combined with blockade of distinct angiogenic pathways to achieve additive to synergistic inhibition of angiogenesis and tumor growth. This will be accomplished using several new adenoviruses designed to antagonize the TIE/angiopoietin and EphB4/ephrin-B2 endothelial receptor tyrosine kinase pathways. Second, we will test the hypothesis that the local and systemic efficacy of oncolytic viruses can be improved by simultaneous treatment with soluble Flk 1 and Flt 1 adenoviruses, or by actually modifying the oncolytic viruses to themselves express anti-angiogenic proteins. Finally, based upon the strong and broad-spectrum activity of the murine FIk 1 and FIt 1 soluble VEGF receptors, we will examine the hypothesis that humanized versions of these proteins will effectively suppress tumor growth and tumor angiogenesis. Moreover, these humanized receptors will be expressed from regulated adenoviruses to confer an additional degree of safety as a prelude to eventual clinical use.